Nearly 70 different autoimmune diseases are known, affecting millions of people worldwide. They involve various organs in the body, such as the joints (eg rheumatoid arthritis; RA), heart, lungs (eg asthma), central nervous system (CNS) (eg multiple sclerosis; MS), endocrine organs (eg type-1 diabetes mellitus; T1DM) and skin (eg psoriasis). Typically, they are characterised by tissue destruction caused by, at least in part, self-reactive T lymphocytes (T cells). As T cells undergo repeated antigen stimulation they differentiate into terminally-differentiated effector memory T (TEM) cells (Sallusto F et al., 2000), which are characterised by high expression of the voltage-gated potassium channel Kv1.3 (after activation) and the absence of both the chemokine receptor CCR7 and phosphatase CD45RA on their surfaces (Wulff H et al., 2003). In the autoimmune diseases mentioned above, the disease-associated T cells in patients with RA (synovial T cells), MS (specific for myelin antigens), T1DM (specific for GAD65 antigens), asthma (induced-sputum T cells) and psoriasis, are all TEM cells (Wulff H et al., 2003; Beeton C et al., 2006; Fasth A et al., 2004; Friedrich M et al., 2000; Koshy S et al., 2014; Lovett-Racke A et al et al., 2003; and Viglietta V et al., 2002). In addition, B cells differentiate into class-switched B cells upon recurring antigen stimulation and are also implicated in MS (Corcione A et al., 2004) and other autoimmune diseases; these cells are a major source of IgG autoantibodies that result in direct tissue damage in RA, MS and T1DM (Berger T et al., 2003; O'Connor K C et al., 2001; Atkinson M A et al., 2001; and Domer T et al., 2003). Similarly to TEM cells, class-switched B cells up-regulate the Kv1.3 potassium channel upon activation and their proliferation can be suppressed through the inhibition of Kv1.3 (Wulff H et al., 2003; and Wulff H et al., 2004). On the other hand, CCR7+ naïve and central memory (TCM) cells are less sensitive to the inhibition of Kv1.3 as they up-regulate KCa3.1 channels upon activation (Wulff H et al., 2003), as do naïve and IgD+CD27+ memory B cells, which are also insensitive to Kv1.3 blocking agents (Wulff H et al., 2003). As a consequence, selective blocking agents of Kv1.3 are expected to reduce the severity of autoimmune diseases without inducing generalised immunosuppression (Beeton C et al., 2011; and Chi Vet al., 2012). Recently, it has also been shown that blocking the Kv1.3 channels has additional therapeutic potential. For example, blocking Kv1.3 with peptides such as ShK, scorpion toxin margatoxin (MgTX) and charybdotoxin (ChTX), prevents the proliferation of CD8+ cytotoxic effector memory T cells and their secretion of granzyme B (GrB), which is toxic to the neuronal cells (Hu L N et al., 2013). These findings indicate that Kv1.3 is not only an attractive therapeutic target for immunomodulation but also plays an important role in neuron protection.
One of the most potent inhibitors of Kv1.3 channels is the sea anemone peptide ShK, which blocks Kv1.3 with an IC50 of 11 pM (Kalman K et al., 1998). ShK is a 35-residue polypeptide consisting of two short α-helices comprising amino acids 14-19 and 21-24 stabilised by three disulphide bridges (Tudor J E et al., 1996). ShK interacts with all four subunits of the Kv1.3 channel tetramer, with Lys22 occluding the channel pore (Kalman K et al., 1998). ShK has been shown to suppress proliferation of TEM cells and improve the condition of two animal models of MS (ie chronic relapse-remitting experimental autoimmune encephalomyelitis (CR-EAE) and adoptive transfer of experimental autoimmune encephalomyelitis (at-EAE) (Beeton C et al., 2006)), the pristane-induced arthritis (PIA) model of RA, and animal models of asthma and psoriasis (Koshy S et al., 2014; and Gilhar A et al., 2011). However, while ShK has considerable therapeutic potential, unfortunately it also binds to the closely-related Kv1 channel subtype, Kv1.1 (Kd=16 pM) that is found in the CNS and heart (Gutman G A et al., 2005). Since it has been shown that Kv1.1-deficient mice exhibit cardiac dysfunction associated with epileptic activity (Glasscock E et al., 2010), there is a need for Kv1.3-selective analogues to be developed in order to avoid potential cardiac- and neuro-toxicity, especially in subjects with MS whose blood-brain barrier (BBB) is disrupted or compromised such that exogenous peptides and proteins may gain entry into the CNS (Bennett J et al., 2010).
Several analogues of ShK with enhanced Kv1.3-selectivity have been synthesised. However, many of these previous analogues included amino acid substitutions with unnatural (ie non-canonical) amino acids and/or non-protein extensions to their N-terminal (Kalman K et al., 1998; Beeton C et al., 2003; and Pennington M W et al., 2009). One such analogue of ShK, known as ShK-186, has recently entered clinical trials for the treatment of a range of autoimmune diseases. This analogue contains an N-terminal phosphotyrosine (pTyr) and a C-terminal amide; the latter was introduced to avoid carboxypeptidase degradation and has no effect on binding affinity (Tarcha E J et al., 2012). ShK-186 is, however, not wholly satisfactory since it is rapidly dephosphorylated in vivo (Tarcha E J et al., 2012) and, further, induces low titre anti-ShK-186 antibody production (Beeton C et al., 2003). Another ShK analogue, known as ShK-192, differs from ShK-186 by the substitution of a methionine (Met21) with norleucine (Nle) to reduce the potential for oxidative metabolism, and the replacement of the phospho moiety with a non-hydrolysable phosphono group. It has been found that while ShK-192 has a slightly lower binding affinity for Kv1.3 channels, it shows a significantly improved level of selectivity over Kv1.1; it is predicted to bind to the extracellular face of the channel with the terminal negatively-charged phosphono group forming a salt bridge with the side-chain ammonium group of Lys411 in Kv1.3 (Pennington M W et al., 2009).
In work leading to the present invention, the applicants employed computational techniques to investigate potential modifications to the ShK toxin to improve selectivity for Kv1.3 channels over Kv1.1 channels. The modifications that were investigated included an N-terminal extension of ShK with the tetrapeptide sequence ESSS (SEQ ID NO: 1) based upon a hypothesis that this extension could mimic the phosphono moiety of the ShK-192 analogue. Molecular dynamics (MD) simulations subsequently indicated that a tryptophan (Trp) at position −3 of the tetrapeptide would be favourable in forming a stable interaction with Pro377 of Kv1.3, so an ShK analogue including an N-terminal extension of EWSS (SEQ ID NO: 2) was also investigated. ShK analogues with novel N-terminal tetrapeptide extensions were therefore designed and produced; electrophysiology results showed that the analogue [EWSS]ShK retains potency against Kv1.3 with an IC50 of 34 pM, but shows a markedly higher level of selectivity for Kv1.3 channels over Kv1.1 channels. These results indicate that the [EWSS]ShK analogue and related analogues may be suitable for use as, for example, therapeutic agents for treating autoimmune disease.